A biomarker of gastric signet ring cell carcinoma and a detection kit thereof

Abstract

The application discloses a biomarker of gastric signet ring cell carcinoma and a detection kit thereof, belongs to the technical field of biological medicine, and particularly relates to a detection kit of a biomarker of gastric signet ring cell carcinoma, total RNA extraction reagent, RNA purification reagent, cDNA first strand synthesis premix reagent, rapid fluorescent quantitative premix reagent (SYBR Green), nuclease-free double-distilled water and specific primers. The RNA purification reagent at least comprises isopropyl alcohol, chloroform and anhydrous ethanol. The specific primers comprise upstream specific primers and downstream specific primers. The application is a detection kit of a biomarker of gastric signet ring cell carcinoma with high RNA purification recovery efficiency and excellent specificity.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a biomarker for gastric signet ring cell carcinoma and its detection kit. Background Technology

[0002] Gastric cancer is one of the most common malignant tumors worldwide and a leading cause of cancer-related deaths. Epidemiological studies show that the overall incidence of gastric cancer, including intestinal-type gastric cancer, is declining year by year, while the incidence of diffuse gastric cancer, especially gastric signet ring cell carcinoma (GSRCC), is increasing year by year. GSRCC develops relatively slowly in its early stages and is generally more indolent than other types of gastric cancer, with its invasiveness only becoming apparent after infiltrating the submucosa. However, once it breaks through the submucosa, GSRCC spreads rapidly and widely, accompanied by distant metastasis and pelvic implantation, exhibiting strong invasiveness, high malignancy, and rapid disease progression. Compared with other types of gastric cancer, GSRCC has a better prognosis in its early stages but a poorer prognosis in its later stages. Therefore, providing a biomarker PTGS2 for the GSRCC population and constructing a prognostic assessment model for GSRCC patients has application value as a potential drug target. Summary of the Invention

[0003] The purpose of this invention is to provide a biomarker for gastric signet ring cell carcinoma and its detection kit, improve RNA purification and recovery efficiency, and optimize the specificity of the detection kit for gastric signet ring cell carcinoma biomarker.

[0004] The technical solution adopted by the present invention to achieve the above objectives is as follows:

[0005] A detection kit for biomarkers of gastric signet ring cell carcinoma includes: total RNA extraction reagent, RNA purification reagent, cDNA first-strand synthesis premix reagent, rapid quantitative PCR premix reagent (SYBR Green), nuclease-free double-distilled water, and specific primers. The RNA purification reagent includes at least isopropanol, chloroform, and anhydrous ethanol. The specific primers include an upstream specific primer and a downstream specific primer, wherein the nucleotide sequence of the upstream specific primer is 5'-TTCTCGTGAAGCCCTATGA-3', and the nucleotide sequence of the downstream specific primer is 5'-GAGGCAGTGTTGATGATTTT-3'. This invention provides a detection kit for biomarkers of gastric signet ring cell carcinoma, which can specifically detect the expression of biomarkers of gastric signet ring cell carcinoma and helps to construct a prognostic assessment model for gastric signet ring cell carcinoma.

[0006] Preferably, the biomarker for gastric signet ring cell carcinoma is prostaglandin intraepoxide synthase 2.

[0007] Preferably, the RNA purification reagent includes isopropanol, chloroform, anhydrous ethanol, and magnetic bead suspension.

[0008] More preferably, in the preparation of the magnetic bead suspension, oleic acid-modified iron oxide magnetic particles are first reacted with ethyl silicate under the action of ammonia, and then reacted with diethylenetriaminepropyltrimethoxysilane and 5-chloro,4-methylvaleroyl chloride. After magnetic separation, ethanol solution is added and ultrasonically dispersed to obtain the magnetic bead suspension. This invention uses diethylenetriaminepropyltrimethoxysilane and 5-chloro,4-methylvaleroyl chloride to modify magnetic beads, introducing a large number of active groups, which helps adsorb nucleic acids, thereby efficiently purifying RNA and improving RNA purification and recovery efficiency. The purified RNA is reverse transcribed into cDNA, and then qRT-PCR is performed using the cDNA as a template, reducing the influence of impurities on qRT-PCR and helping to improve the specificity of the detection kit for gastric signet ring cell carcinoma biomarkers of this invention.

[0009] More preferably, the ratio of oleic acid-modified iron oxide magnetic particles to ammonia is 1g:5-20mL.

[0010] More preferably, the ratio of oleic acid-modified magnetite particles to ethyl silicate is 1g:2-10mL.

[0011] More preferably, the ratio of oleic acid-modified iron oxide magnetic particles to diethylenetriaminepropyltrimethoxysilane is 1g:2-10mL.

[0012] More preferably, the mass ratio of oleic acid-modified magnetite particles and 5-chloro,4-methylpentanoyl chloride is 1:2-10.

[0013] More preferably, the mass concentration of the ethanol solution is 75-95%.

[0014] More preferably, the ratio of oleic acid-modified iron oxide magnetic particles to ethanol solution is 1g:50-150mL.

[0015] More preferably, the preparation of the magnetic bead suspension specifically involves,

[0016] Under a nitrogen atmosphere, oleic acid-modified iron oxide magnetic particles were added to deionized water and stirred to disperse them. Ammonia water was added and stirred to disperse them again. Ethyl silicate was added dropwise and the reaction was stirred at 35-50℃ for 1-3 hours. After the reaction was completed, the mixture was naturally cooled to room temperature and washed with anhydrous ethanol until the pH was neutral. After magnetic separation, ethanol solution was added and ultrasonically dispersed to obtain a magnetic bead suspension.

[0017] More preferably, the preparation of the magnetic bead suspension specifically involves,

[0018] Under a nitrogen atmosphere, oleic acid-modified iron oxide magnetic particles were added to deionized water and stirred to disperse them. Ammonia water was added and stirred to disperse them again. Ethyl silicate was added dropwise, and the reaction was stirred at 35-50℃ for 1-3 hours. After the reaction was completed, the mixture was naturally cooled to room temperature. Ammonia water was added to adjust the pH to 9-10, and diethylenetriaminepropyltrimethoxysilane was added and reacted for 4-8 hours. 5-chloro,4-methylpentanoyl chloride and triethylamine were added at 0-5℃, and the reaction was stirred at room temperature for 3-6 hours. The mixture was washed with anhydrous ethanol until the pH was neutral. After magnetic separation, ethanol solution was added and ultrasonically dispersed to obtain a magnetic bead suspension.

[0019] More preferably, the preparation of the magnetic bead suspension specifically involves,

[0020] Under a nitrogen atmosphere, oleic acid-modified magnetite (Fe3O4) magnetic particles were added to deionized water and stirred to disperse. Ammonia was added, and the mixture was stirred again. Ethyl silicate was added dropwise, and the reaction was carried out at 35-50°C with stirring for 1-3 hours. After the reaction, the mixture was allowed to cool naturally to room temperature. Ammonia was added to adjust the pH to 9-10, and diethylenetriaminepropyltrimethoxysilane was added and reacted for 4-8 hours. 5-chloro,4-methylpentanoyl chloride and triethylamine were added at 0-5°C, and the reaction was carried out at room temperature with stirring for 3-6 hours. N-(tert-Butoxycarbonyl)ethanolamine, anhydrous potassium carbonate, and potassium iodide were then added, and the reaction was carried out at 70-80°C for 2-6 hours. After the reaction, the mixture was allowed to cool naturally to room temperature, washed with anhydrous ethanol until the pH was neutral, and after magnetic separation, an ethanol solution was added and ultrasonically dispersed to obtain a magnetic bead suspension. This invention further utilizes N-(tert-Butoxycarbonyl)ethanolamine to modify the magnetic beads, which helps to modify the surface of the magnetic beads with active groups, further improving the efficiency of RNA purification and recovery, thereby further improving the specificity of the detection kit for gastric signet ring cell carcinoma biomarkers.

[0021] More preferably, the ratio of oleic acid-modified iron oxide magnetic particles to deionized water is 1g:50-150mL.

[0022] More preferably, the ratio of oleic acid-modified iron oxide magnetic particles to ammonia is 1g:5-20mL.

[0023] More preferably, the ratio of oleic acid-modified magnetite particles to ethyl silicate is 1g:2-10mL.

[0024] More preferably, the ratio of oleic acid-modified iron oxide magnetic particles to diethylenetriaminepropyltrimethoxysilane is 1g:2-10mL.

[0025] More preferably, the mass ratio of oleic acid-modified magnetite particles and 5-chloro,4-methylpentanoyl chloride is 1:2-10.

[0026] More preferably, the ratio of 5-chloro,4-methylpentanoyl chloride to triethylamine is 1 g: 0.1-0.2 mL.

[0027] More preferably, the mass ratio of oleic acid-modified magnetite particles to N-(tert-butyloxycarbonyl)ethanolamine is 1:5-20.

[0028] More preferably, the mass ratio of oleic acid-modified magnetite particles to anhydrous potassium carbonate is 1:1-2.

[0029] More preferably, the mass ratio of oleic acid-modified magnetite particles to potassium iodide is 1:0.1-0.5.

[0030] More preferably, the mass concentration of the ethanol solution is 75-95%, and the ratio of oleic acid-modified iron oxide magnetic particles to ethanol solution is 1g:50-150mL.

[0031] Preferably, the detection kit for biomarkers of gastric signet ring cell carcinoma includes,

[0032] 50-100 parts total RNA extraction reagent, 20-80 parts isopropanol, 10-40 parts chloroform, 60-240 parts anhydrous ethanol, 250-1000 parts nuclease-free double-distilled water, 11-44 parts magnetic bead suspension, 0.2-0.8 parts cDNA first-strand synthesis premix reagent, 2-10 parts rapid real-time fluorescence premix reagent (SYBR Green), 0.05-0.3 parts upstream specific primer and 0.05-0.3 parts downstream specific primer.

[0033] This invention also discloses a method for detecting biomarkers of gastric signet ring cell carcinoma, comprising,

[0034] S1. After adding total RNA extraction reagent to the tissue, homogenize the mixture and incubate at room temperature for 3-6 min. Centrifuge at 10000-12000 rpm for 5-15 min at 0-5℃. Collect the supernatant, add chloroform, vortex for 10-20 s, incubate at room temperature for 2-4 min, centrifuge at 10000-12000 rpm for 10-20 min at 0-5℃, collect the aqueous phase, add isopropanol and mix well. Incubate at room temperature for 5-15 min, centrifuge at 10000-12000 rpm for 5-15 min at 0-5℃, remove the supernatant, add ethanol solution, centrifuge at 10000-12000 rpm for 5-10 min at 0-5℃, remove the waste liquid, air dry for 2-5 min, add nuclease-free double-distilled water, and repeatedly pipette and mix to obtain the RNA solution.

[0035] S2. On a magnetic rack, mix the RNA solution and magnetic bead suspension evenly, let stand at room temperature for 2-10 min, centrifuge briefly, let stand for 2-10 min, add ethanol solution, pipette 2-5 times, let stand for 1-5 min, remove the supernatant, repeat adding ethanol solution, pipette 2-5 times, let stand for 1-5 min, remove the supernatant, then air dry at room temperature for 2-10 min, add nuclease-free double-distilled water, gently pipette 2-5 times, let stand for 2-10 min, to obtain the purified RNA solution.

[0036] S3. Dilute the purified RNA solution with nuclease-free double-distilled water to a final concentration of 10-100 ng / μL to obtain the RNA template solution; take the RNA template solution, cDNA first-strand synthesis premix reagent and nuclease-free double-distilled water, react at 40-50℃ for 10-20 min, and then react at 94-97℃ for 2-5 min to obtain the cDNA solution.

[0037] S4. Dilute the cDNA solution with nuclease-free double-distilled water to a final concentration of 20-100 ng / μL to obtain the cDNA dilution. Take the rapid real-time quantitative reagent (SYBR Green), upstream specific primer, downstream specific primer, cDNA dilution, and nuclease-free double-distilled water, and use a qRT-PCR instrument to detect the expression level of the target gene. The qRT-PCR reaction system is as follows: react at 94-97℃ for 1-3 min, then react at 94-97℃ for 5-10 s, 50-60℃ for 10-15 s, and 70-75℃ for 10-20 s for 34-40 cycles.

[0038] Preferably, the ratio of tissue and total RNA extraction reagent used in step S1 is 1g:20-40mL.

[0039] Preferably, the volume ratio of chloroform to total RNA extraction reagent in step S1 is 1:2-10.

[0040] Preferably, the volume ratio of isopropanol to total RNA extraction reagent in step S1 is 1:1-2.

[0041] Preferably, in the preparation of the ethanol solution in step S1, anhydrous ethanol is diluted with nuclease-free ultrapure water to a mass concentration of 70-90%.

[0042] Preferably, the volume ratio of total RNA extraction reagent to ethanol solution in step S1 is 1:1-2.

[0043] Preferably, the volume ratio of total RNA extraction reagent to nuclease-free double-distilled water in step S1 is 1:0.1-0.2.

[0044] Preferably, the volume ratio of RNA solution to magnetic bead suspension in step S2 is 1:2-4.

[0045] Preferably, in the preparation of the ethanol solution in step S2, anhydrous ethanol is diluted with nuclease-free ultrapure water to a mass concentration of 70-90%.

[0046] Preferably, the volume ratio of RNA solution to ethanol solution in step S2 is 1:2-4.

[0047] Preferably, in step S2, the volume ratio of RNA solution to nuclease-free double-distilled water is 1:1-2.

[0048] Preferably, in step S3, the volume ratio of the RNA template solution to the cDNA first-strand synthesis premix reagent is 1:4-8.

[0049] Preferably, in step S3, the volume ratio of RNA template solution to nuclease-free double-distilled water is 1:5-20.

[0050] Preferably, in step S4, the volume ratio of cDNA diluent to upstream specific primer is 1:0.2-1.

[0051] Preferably, in step S4, the volume ratio of cDNA diluent to downstream specific primer is 1:0.2-1.

[0052] Preferably, in step S4, the volume ratio of cDNA diluent to rapid real-time quantitative premixed reagent (SYBR Green) is 1:5-20.

[0053] Preferably, in step S4, the volume ratio of cDNA diluent to nuclease-free double-distilled water is 1:5-10.

[0054] This invention utilizes oleic acid-modified magnetite (Fe3O4) magnetic particles, which are first reacted with ethyl silicate, and then with diethylenetriaminepropyltrimethoxysilane, 5-chloro,4-methylpentanoyl chloride, and N-(tert-butyloxycarbonyl)ethanolamine to obtain a magnetic bead suspension. This magnetic bead suspension is then used to compose a detection kit for gastric signet ring cell carcinoma biomarkers. Therefore, this invention offers the following advantages: the magnetic bead suspension in the gastric signet ring cell carcinoma biomarker detection kit efficiently purifies RNA, with an RNA purification and recovery efficiency of 85.34-98.73%. High-purity RNA is reverse transcribed into cDNA, and then qRT-PCR is performed using the cDNA as a template, reducing the influence of impurities on qRT-PCR and thus improving the specificity of the gastric signet ring cell carcinoma biomarker detection kit, with a specificity of 88.64-99.77%. Therefore, this invention provides a detection kit for gastric signet ring cell carcinoma biomarkers with high RNA purification and recovery efficiency and excellent specificity. Attached Figure Description

[0055] Figure 1 The image shows a TEM image of the magnetic bead suspension prepared in Example 2.

[0056] Figure 2 This shows the mRNA expression status of PTGS2.

[0057] Figure 3 This shows the protein expression status of PTGS2.

[0058] Figure 4 To investigate the migration of gastric signet ring cancer cells after PTGS2 knockdown.

[0059] Figure 5 To reduce the invasion of gastric signet ring cell carcinoma cells after PTGS2 knockdown.

[0060] Figure 6 To reduce the adhesion of gastric signet ring cancer cells after PTGS2 knockdown.

[0061] Figure 7 To determine the plate colony status of gastric signet ring cell carcinoma cells after PTGS2 knockdown.

[0062] Figure 8 To investigate the proliferation of gastric signet ring cell carcinoma cells after PTGS2 knockdown.

[0063] Figure 9 To knock down the soft agar colony-forming ability of gastric signet ring cell carcinoma cells after PTGS2.

[0064] Figure 10 The migration of gastric signet ring cancer cells after celecoxib treatment.

[0065] Figure 11 The proliferation of gastric signet ring cancer cells after celecoxib treatment.

[0066] Figure 12 To investigate the migration of gastric signet ring cancer cells after PTGS2 overexpression.

[0067] Figure 13 To investigate the invasion of gastric signet ring cancer cells after overexpression of PTGS2.

[0068] Figure 14 To investigate the adhesion of gastric signet ring cancer cells after PTGS2 overexpression.

[0069] Figure 15 To investigate the proliferation of gastric signet ring cancer cells after overexpression of PTGS2.

[0070] Figure 16 To assess the soft agar colony-forming ability of gastric signet ring cell carcinoma cells after overexpression of PTGS2.

[0071] Figure 17 The expression of adhesion molecules after overexpression of PTGS2.

[0072] Figure 18 To investigate the mRNA expression of IQGAP1 in gastric signet ring cell carcinoma cells after PTGS2 knockdown.

[0073] Figure 19 To determine the mRNA expression of IQGAP1 in gastric signet ring cell carcinoma cells after PTGS2 overexpression.

[0074] Figure 20 To investigate the protein expression of IQGAP1 in gastric signet ring cell carcinoma cells after PTGS2 knockdown.

[0075] Figure 21 The protein expression of IQGAP1 in gastric signet ring cell carcinoma cells after celecoxib treatment.

[0076] Figure 22 To determine the protein expression of IQGAP1 in gastric signet ring cell carcinoma cells after PTGS2 overexpression.

[0077] Figure 23 To investigate the protein expression of PTGS2 in gastric signet ring cell carcinoma cells after knocking down IQGAP1.

[0078] Figure 24 To investigate the migration of gastric signet ring cell carcinoma cells after knocking down IQGAP1.

[0079] Figure 25 To reduce the invasion of gastric signet ring cell carcinoma cells after knocking down IQGAP1.

[0080] Figure 26 To reduce the adhesion of gastric signet ring cells to IQGAP1.

[0081] Figure 27 To determine the soft agar clone status of gastric signet ring cell carcinoma cells after IQGAP1 knockdown.

[0082] Figure 28 Correlation analysis of mRNA expression levels of PTGS2 and IQGAP1.

[0083] Figure 29 This study investigated the mRNA expression of ERK target genes and cell cycle molecules in gastric signet ring cell carcinoma after PTGS2 overexpression.

[0084] Figure 30 To investigate the mRNA expression of ERK target genes and cell cycle molecules in gastric signet ring cell carcinoma after PTGS2 knockdown.

[0085] Figure 31 To knock down the protein expression level of MMP2 in gastric signet ring cell carcinoma cells after PTGS2.

[0086] Figure 32To knock down the protein expression level of MMP2 in gastric signet ring cell carcinoma cells after IQGAP1.

[0087] Figure 33 To knock down the phosphorylation level of the ERK pathway in gastric signet ring cell carcinoma cells after IQGAP1.

[0088] Figure 34 To determine the phosphorylation level of the ERK pathway in gastric signet ring cell carcinoma cells after PTGS2 overexpression. Detailed Implementation

[0089] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0090] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0091] Example 1:

[0092] A biomarker for gastric signet ring cell carcinoma includes prostaglandin-endoperoxide synthase 2 (PTGS2).

[0093] Preparation of magnetic bead suspension, including,

[0094] Under a nitrogen atmosphere, oleic acid-modified magnetite (Fe3O4) magnetic particles were added to deionized water and stirred to disperse. Ammonia was added, and the mixture was stirred again to disperse. Ethyl silicate was added dropwise, and the reaction was carried out at 40°C with stirring for 2 hours. After the reaction, the mixture was allowed to cool naturally to room temperature. The particles were washed with anhydrous ethanol until the pH was neutral. After magnetic separation, an ethanol solution was added and ultrasonically dispersed to obtain a magnetic bead suspension. The volume ratio of oleic acid-modified magnetite (Fe3O4) magnetic particles to deionized water was 1 g:100 mL; the volume ratio of oleic acid-modified magnetite (Fe3O4) magnetic particles to ammonia was 1 g:10 mL; the volume ratio of oleic acid-modified magnetite (Fe3O4) magnetic particles to ethyl silicate was 1 g:5 mL; the ethanol solution had a mass concentration of 85%, and the volume ratio of oleic acid-modified magnetite (Fe3O4) magnetic particles to ethanol solution was 1 g:100 mL.

[0095] A detection kit for biomarkers of gastric signet ring cell carcinoma, comprising,

[0096] 100 parts by volume of total RNA extraction reagent, 40 parts by volume of isopropanol, 20 parts by volume of chloroform, 120 parts by volume of anhydrous ethanol, 500 parts by volume of nuclease-free double-distilled water, 22 parts by volume of magnetic bead suspension, 0.4 parts by volume of cDNA first-strand synthesis premix reagent, 5 parts by volume of rapid quantitative PCR premix reagent (SYBR Green), 0.15 parts by volume of upstream specific primers, and 0.15 parts by volume of downstream specific primers. The total RNA extraction reagent, nuclease-free double-distilled water, cDNA first-strand synthesis premix reagent, and rapid quantitative PCR premix reagent (SYBR Green) were purchased from Tiangen Biotech (Beijing) Co., Ltd. The ethanol solution was obtained by diluting anhydrous ethanol with nuclease-free double-distilled water to a final concentration of 75%. The upstream and downstream specific primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The nucleotide sequence of the upstream specific primer is shown in SEQ ID No. 1, which is 5'-TTCTCGTGAAGCCCTATGA-3'; the nucleotide sequence of the downstream specific primer is shown in SEQ ID No. 2, which is 5'-GAGGCAGTGTTGATGATTTT-3'.

[0097] A method for detecting biomarkers of gastric signet ring cell carcinoma, comprising,

[0098] S1. After adding total RNA extraction reagent to the tissue, homogenize the mixture and let it stand at room temperature for 5 min. Centrifuge at 10,000 rpm for 10 min at 4℃, collect the supernatant, add chloroform, vortex for 15 s, let it stand at room temperature for 3 min, centrifuge at 10,000 rpm for 15 min at 4℃, collect the aqueous phase, add isopropanol and mix well, let it stand at room temperature for 10 min, centrifuge at 10,000 rpm for 10 min at 4℃, remove the supernatant, add ethanol solution, centrifuge at 10,000 rpm for 5 min at 4℃, remove the waste liquid, air dry for 3 min, add nuclease-free double-distilled water, and repeatedly pipette and mix to obtain the RNA solution. The volume ratio of tissue and total RNA extraction reagent was 1 g: 33 mL; the volume ratio of chloroform and total RNA extraction reagent was 1:5; the volume ratio of isopropanol and total RNA extraction reagent was 1:2; in the preparation of the ethanol solution, anhydrous ethanol was diluted with nuclease-free ultrapure water to a mass concentration of 75%; the volume ratio of total RNA extraction reagent to ethanol solution was 1:1; and the volume ratio of total RNA extraction reagent to nuclease-free double-distilled water was 1:0.1.

[0099] S2. On a magnetic rack, mix the RNA solution and magnetic bead suspension thoroughly, let stand at room temperature for 5 min, centrifuge briefly, let stand for 5 min, add ethanol solution, pipette 5 times, let stand for 2 min, remove the supernatant, repeat adding ethanol solution, pipette 5 times, let stand for 2 min, remove the supernatant, then air dry at room temperature for 5 min, add nuclease-free double-distilled water, gently pipette 5 times, let stand for 5 min, to obtain the purified RNA solution. The volume ratio of RNA solution to magnetic bead suspension is 1:2; in the preparation of ethanol solution, use nuclease-free ultrapure water to dilute anhydrous ethanol to a mass concentration of 80%; the volume ratio of RNA solution to ethanol solution is 1:2; the volume ratio of RNA solution to nuclease-free double-distilled water is 1:1.

[0100] S3. Dilute the purified RNA solution with nuclease-free double-distilled water to a final concentration of 50 ng / μL to obtain the RNA template solution. Take the RNA template solution, cDNA first-strand synthesis premix reagent, and nuclease-free double-distilled water, and react at 45℃ for 15 min, then at 95℃ for 3 min to obtain the cDNA solution. The volume ratio of RNA template solution to cDNA first-strand synthesis premix reagent is 1:4; the volume ratio of RNA template solution to nuclease-free double-distilled water is 1:15.

[0101] S4. Dilute the cDNA solution to a final concentration of 50 ng / μL with nuclease-free double-distilled water to obtain the cDNA dilution buffer. Take 10 μL of SYBR Green rapid real-time quantitative reagent, 0.6 μL of upstream specific primer, 0.6 μL of downstream specific primer, 1 μL of cDNA dilution buffer, and 7.8 μL of nuclease-free double-distilled water. Use a qRT-PCR instrument to detect the expression level of the target gene. The qRT-PCR reaction system is: 95℃ for 1 min, followed by 40 cycles of 95℃ for 5 s, 60℃ for 10 s, and 72℃ for 15 s. The volume ratio of cDNA dilution buffer to upstream specific primer is 1:0.6; the volume ratio of cDNA dilution buffer to downstream specific primer is 1:0.6; the volume ratio of cDNA dilution buffer to SYBR Green rapid real-time quantitative reagent is 1:10; and the volume ratio of cDNA dilution buffer to nuclease-free double-distilled water is 1:7.8.

[0102] Example 2:

[0103] A biomarker for gastric signet ring cell carcinoma, same as in Example 1.

[0104] Preparation of magnetic bead suspension, including,

[0105] Oleic acid-modified iron oxide magnetic particles were added to deionized water and stirred to disperse under a nitrogen atmosphere. Ammonia was added and the mixture was stirred to disperse again. Ethyl silicate was added dropwise and the mixture was stirred and reacted at 40°C for 2 hours. After the reaction was completed, the mixture was naturally cooled to room temperature. Ammonia was added to adjust the pH to 10. Diethylenetriaminepropyltrimethoxysilane was added and the mixture was reacted for 6 hours. 5-Chloro,4-methylpentanoyl chloride and triethylamine were added at 0°C and the mixture was stirred and reacted at room temperature for 5 hours. The mixture was washed with anhydrous ethanol until the pH was neutral. After magnetic separation, ethanol solution was added and the mixture was ultrasonically dispersed to obtain a magnetic bead suspension. The ratio of oleic acid-modified magnetite (Fe3O4) magnetic particles to deionized water was 1 g: 100 mL; the ratio of oleic acid-modified magnetite (Fe3O4) magnetic particles to ammonia was 1 g: 10 mL; the ratio of oleic acid-modified magnetite (Fe3O4) magnetic particles to ethyl silicate was 1 g: 5 mL; the ratio of oleic acid-modified magnetite (Fe3O4) magnetic particles to diethylenetriaminepropyltrimethoxysilane was 1 g: 5 mL; the mass ratio of oleic acid-modified magnetite (Fe3O4) magnetic particles to 5-chloro,4-methylpentanoyl chloride was 1:5; the ratio of 5-chloro,4-methylpentanoyl chloride to triethylamine was 1 g: 0.15 mL; the mass concentration of the ethanol solution was 85%, and the ratio of oleic acid-modified magnetite (Fe3O4) magnetic particles to the ethanol solution was 1 g: 100 mL.

[0106] A detection kit for gastric signet ring cell carcinoma biomarkers, which is the same as Example 1 except that the magnetic bead suspension is replaced with the magnetic bead suspension prepared in this example.

[0107] A method for detecting biomarkers of gastric signet ring cell carcinoma, which is the same as in Example 1 except that the magnetic bead suspension is replaced with the magnetic bead suspension prepared in this example.

[0108] Example 3:

[0109] A biomarker for gastric signet ring cell carcinoma, same as in Example 1.

[0110] The preparation of the magnetic bead suspension is the same as in Example 2.

[0111] A detection kit for gastric signet ring cell carcinoma biomarkers, which is the same as Example 1 except that the magnetic bead suspension is replaced with the modified magnetic bead suspension prepared in this example.

[0112] A method for detecting biomarkers of gastric signet ring cell carcinoma, compared with Example 1, except that in step S2 the magnetic bead suspension is replaced with the modified magnetic bead suspension prepared in this example, and the volume ratio of RNA solution to magnetic bead suspension is changed to 1:1, all other conditions are the same as in Example 2.

[0113] Example 4:

[0114] A biomarker for gastric signet ring cell carcinoma, same as in Example 1.

[0115] Preparation of magnetic bead suspension, including,

[0116] Under a nitrogen atmosphere, oleic acid-modified iron oxide magnetic particles were added to deionized water and stirred to disperse. Ammonia was added and stirred to disperse again. Ethyl silicate was added dropwise, and the reaction was stirred at 40°C for 2 hours. After the reaction was completed, the mixture was allowed to cool to room temperature. Ammonia was added to adjust the pH to 10, and diethylenetriaminepropyltrimethoxysilane was added and reacted for 6 hours. 5-chloro,4-methylpentanoyl chloride and triethylamine were added at 0°C and stirred at room temperature for 5 hours. N-(tert-butyloxycarbonyl)ethanolamine, anhydrous potassium carbonate, and potassium iodide were then added and reacted at 70°C for 5 hours. After the reaction was completed, the mixture was allowed to cool to room temperature, washed with anhydrous ethanol until the pH was neutral, and after magnetic separation, an ethanol solution was added and ultrasonically dispersed to obtain a magnetic bead suspension. The ratio of oleic acid-modified magnetite (Fe3O4) magnetic particles to deionized water was 1 g: 100 mL; the ratio of oleic acid-modified magnetite (Fe3O4) magnetic particles to ammonia was 1 g: 10 mL; the ratio of oleic acid-modified magnetite (Fe3O4) magnetic particles to ethyl silicate was 1 g: 5 mL; the ratio of oleic acid-modified magnetite (Fe3O4) magnetic particles to diethylenetriaminepropyltrimethoxysilane was 1 g: 5 mL; the mass ratio of oleic acid-modified magnetite (Fe3O4) magnetic particles to 5-chloro,4-methylpentanoyl chloride was 1:5; 5 The mass ratio of 4-methylpentanoyl chloride to triethylamine is 1 g: 0.15 mL; the mass ratio of oleic acid-modified magnetite particles to N-(tert-butyloxycarbonyl)ethanolamine is 1:10; the mass ratio of oleic acid-modified magnetite particles to anhydrous potassium carbonate is 1:1; the mass ratio of oleic acid-modified magnetite particles to potassium iodide is 1:0.1; the mass concentration of the ethanol solution is 85%, and the mass ratio of oleic acid-modified magnetite particles to ethanol solution is 1 g: 100 mL.

[0117] A detection kit for gastric signet ring cell carcinoma biomarkers, which is the same as Example 1 except that the magnetic bead suspension is replaced with the modified magnetic bead suspension prepared in this example.

[0118] A method for detecting biomarkers of gastric signet ring cell carcinoma, compared with Example 1, except that in step S2 the magnetic bead suspension is replaced with the modified magnetic bead suspension prepared in this example, all other conditions are the same as in Example 1.

[0119] Example 5:

[0120] A biomarker for gastric signet ring cell carcinoma, same as in Example 1.

[0121] The preparation of the magnetic bead suspension is the same as in Example 4.

[0122] A detection kit for gastric signet ring cell carcinoma biomarkers, which is the same as Example 1 except that the magnetic bead suspension is replaced with the modified magnetic bead suspension prepared in this example.

[0123] A method for detecting biomarkers of gastric signet ring cell carcinoma, compared with Example 1, except that in step S2 the magnetic bead suspension is replaced with the modified magnetic bead suspension prepared in this example, and the volume ratio of RNA solution to magnetic bead suspension is changed to 1:1, all other conditions are the same as in Example 1.

[0124] Comparative Example 1:

[0125] A biomarker for gastric signet ring cell carcinoma, same as in Example 1.

[0126] The preparation of the magnetic bead suspension was the same as in Example 4, except that 5-chloro,4-methylpentanoyl chloride was not added.

[0127] A detection kit for gastric signet ring cell carcinoma biomarkers, which is the same as Example 1 except that the magnetic bead suspension is replaced with the modified magnetic bead suspension prepared in this comparative example.

[0128] A method for detecting biomarkers of gastric signet ring cell carcinoma, compared with Example 1, except that in step S2 the magnetic bead suspension is replaced with the modified magnetic bead suspension prepared in this comparative example, all other conditions are the same as in Example 1.

[0129] Comparative Example 2:

[0130] A biomarker for gastric signet ring cell carcinoma, same as in Example 1.

[0131] The preparation of the magnetic bead suspension was the same as in Example 4, except that diethylenetriaminepropyltrimethoxysilane and 5-chloro,4-methylpentanoyl chloride were not added.

[0132] A detection kit for gastric signet ring cell carcinoma biomarkers, which is the same as Example 1 except that the magnetic bead suspension is replaced with the modified magnetic bead suspension prepared in this comparative example.

[0133] A method for detecting biomarkers of gastric signet ring cell carcinoma, compared with Example 1, except that in step S2 the magnetic bead suspension is replaced with the modified magnetic bead suspension prepared in this comparative example, all other conditions are the same as in Example 1.

[0134] Experimental example:

[0135] 1. Material Characterization

[0136] The magnetic bead suspension prepared in Example 2 was characterized by transmission electron microscopy (TEM).

[0137] Figure 1 The image shows a TEM image of the magnetic bead suspension prepared in Example 2. The magnetic bead particles in the magnetic bead suspension prepared in this invention are regular spherical, and there is particle accumulation and aggregation.

[0138] 2. Biological Experiments

[0139] 2.1 Experimental Methods

[0140] Three cell lines were constructed from gastric signet ring cell carcinoma cells: gastric signet ring cell carcinoma cells overexpressing PTGS2, gastric signet ring cell carcinoma cells with PTGS2 knockdown, and gastric signet ring cell carcinoma cells with IQGAP1 knockdown. For the construction of PTGS2-overexpressing gastric signet ring cell carcinoma cells, two treatment groups were set up: a PTGS2 overexpression control group (oe-con) and a PTGS2 overexpression experimental group (oe-PTGS2). For the construction of PTGS2 knockdown gastric signet ring cell carcinoma cells, three treatment groups were set up: a PTGS2 knockdown control group (sh-con), a PTGS2 knockdown experimental group 1 (sh1-PTGS2), and a PTGS2 knockdown experimental group 2 (sh2-PTGS2). For the construction of IQGAP1 knockdown gastric signet ring cell carcinoma cells, three treatment groups were set up: an IQGAP1 knockdown control group (sh-con), an IQGAP1 knockdown experimental group 1 (sh1-IQGAP1), and an IQGAP1 knockdown experimental group 2 (sh2-IQGAP1).

[0141] Celecoxib treatment of gastric signet ring cell carcinoma cells: Celecoxib is a specific inhibitor of PTGS2. Celecoxib was diluted to final concentrations of 0 μmol / L, 20 μmol / L, and 40 μmol / L using sterile ultrapure water. Treatment with celecoxib at these final concentrations was performed for 24 h.

[0142] mRNA expression level detection: The expression level of target gene mRNA was detected by qPT-PCR.

[0143] Protein expression level and pathway phosphorylation level detection: Western blotting was used to detect the protein expression level of the target gene and the phosphorylation level of the pathway.

[0144] Detection of malignant phenotypes in gastric signet ring cell carcinomas: proliferation capacity was detected by CCK-8 assay and plate colony formation assay; motility was detected by Transwell migration and invasion assay; adhesion and colony formation capacity were detected by Matrigel adhesion assay and soft agar colony formation assay.

[0145] 2.2 Expression of PTGS2

[0146] Figure 2 This shows the mRNA expression status of PTGS2. Figure 3 This shows the protein expression status of PTGS2. Figure 2 and Figure 3 In the above, S1 represents the expression level of PTGS2 in paired normal tissues, and S2 represents the expression level of PTGS2 in gastric signet ring cell carcinoma tissues. Figure 2 and Figure 3 It was found that, compared with paired normal tissues, the mRNA and protein expression levels of PTGS2 in gastric signet ring cell carcinoma tissues showed an increasing trend. This indicates that PTGS2 is highly expressed in gastric signet ring cell carcinoma tissues.

[0147] 2.3 Effects of PTGS2 on the malignant phenotype of gastric signet ring cell carcinomas

[0148] Figure 4 To reduce the migration of gastric signet ring cell carcinoma cells after PTGS2 knockdown, Figure 5 To knock down the invasion of gastric signet ring cell carcinoma cells after PTGS2, Figure 6 To reduce the adhesion of gastric signet ring cells to PTGS2, Figure 7 To determine the plate colony composition of gastric signet ring cell carcinoma cells after PTGS2 knockdown, Figure 8 To reduce the proliferation of gastric signet ring cell carcinoma cells after PTGS2 knockdown, Figure 9 To knock down the soft agar colony-forming ability of gastric signet ring cell carcinoma cells after PTGS2. Figures 4 to 9 It is known that knocking down PTGS2 significantly inhibits the migration, invasion, adhesion, plate cloning, proliferation, and soft agar cloning ability of gastric signet ring cell carcinomas.

[0149] Figure 10 The migration of gastric signet ring cell carcinoma cells after celecoxib treatment; Figure 11 The proliferation of gastric signet ring cell carcinoma cells after celecoxib treatment. Figure 10 and Figure 11 It can be seen that the migration and proliferation ability of gastric signet ring cancer cells is significantly reduced after treatment with celecoxib.

[0150] Figure 12 To investigate the migration of gastric signet ring cell carcinomas after PTGS2 overexpression, Figure 13 To investigate the invasion of gastric signet ring cell carcinomas after overexpression of PTGS2, Figure 14 To investigate the adhesion of gastric signet ring cells after PTGS2 overexpression, Figure 15 To investigate the proliferation of gastric signet ring cell carcinomas after overexpression of PTGS2, Figure 16 To assess the soft agar colony-forming ability of gastric signet ring cell carcinoma cells overexpressing PTGS2. Figures 12 to 16It can be seen that overexpression of PTGS2 significantly promotes the migration, invasion, adhesion, proliferation and soft agar colony formation of gastric signet ring cell carcinomas.

[0151] Depend on Figures 4 to 16 It is known that PTGS2 promotes various malignant biological behaviors of gastric signet ring cell carcinoma cells, and the specific inhibitor celecoxib inhibits the malignant phenotype of gastric signet ring cell carcinoma cells.

[0152] 2.4 PTGS2 positively regulates the expression level of IQGAP1

[0153] Figure 17 This describes the expression of adhesion molecules after PTGS2 overexpression. Figure 17 It can be seen that overexpression of PTGS2 in gastric signet ring cell carcinoma cells alters the expression levels of adhesion molecules, with the most significant increase in the mRNA expression level of the adhesion molecule IQGAP1. Figure 18 To determine the mRNA expression of IQGAP1 in gastric signet ring cell carcinoma cells after PTGS2 knockdown, Figure 19 To determine the mRNA expression of IQGAP1 in gastric signet ring cell carcinoma cells after PTGS2 overexpression, Figure 20 To determine the protein expression of IQGAP1 in gastric signet ring cell carcinoma cells after PTGS2 knockdown, Figure 21 The expression of IQGAP1 protein in gastric signet ring cell carcinoma cells after celecoxib treatment. Figure 22 To illustrate the protein expression of IQGAP1 in gastric signet ring cell carcinoma cells after PTGS2 overexpression. Figures 18 to 22 It can be seen that PTGS2 regulates the mRNA and protein expression levels of IQGAP1. Figure 23 To investigate the protein expression of PTGS2 in gastric signet ring cell carcinoma cells after IQGAP1 knockdown. Figure 23 It was found that knocking down IQGAP1 in gastric signet ring cell carcinoma cells did not affect the protein expression level of PTGS2. This indicates that IQGAP1 is a downstream target gene of PTGS2 in gastric signet ring cell carcinoma cells.

[0154] 2.5 Effect of IQGAP1 on the malignant phenotype of gastric signet ring cell carcinomas

[0155] Figure 24 To reduce the migration of gastric signet ring cell carcinoma cells after IQGAP1 knockdown, Figure 25 To reduce the invasion of gastric signet ring cell carcinoma cells after IQGAP1 knockdown, Figure 26 To reduce the adhesion of gastric signet ring cells to IQGAP1, Figure 27 Soft agar colonies of gastric signet ring cell carcinomas after IQGAP1 knockdown. (From...) Figures 24 to 27It can be seen that knocking down IQGAP1 weakens the migration, invasion, adhesion, and soft agar cloning ability of gastric signet ring cell carcinoma cells, indicating that IQGAP1 promotes various malignant biological behaviors of gastric signet ring cell carcinoma cells.

[0156] Figure 28 Correlation analysis of PTGS2 and IQGAP1 mRNA expression levels showed a significant correlation between them, with a correlation coefficient (R) of 0.55. This indicates that PTGS2 promotes the malignant phenotype of gastric signet ring cell carcinoma by upregulating IQGAP1 expression.

[0157] 2.6 Exploring the potential downstream mechanisms by which the PTGS2-IQGAP1 axis promotes the malignant phenotype of gastric signet ring cell carcinoma

[0158] Figure 29 To determine the mRNA expression of ERK target genes and cell cycle molecules in gastric signet ring cell carcinoma after PTGS2 overexpression. Figure 30 To investigate the mRNA expression of ERK target genes and cell cycle molecules in gastric signet ring cell carcinoma after PTGS2 knockdown. Figure 29 and Figure 30 It was found that overexpression of PTGS2 significantly increased the mRNA expression levels of ERK target genes and cell cycle molecules, including MMP2, c-myc, and N-cadheirn, in gastric signet ring cell carcinoma cells; knockdown of PTGS2 in gastric signet ring cell carcinoma cells significantly inhibited the mRNA expression levels of some cell cycle molecules and ERK target genes; among them, the change in MMP2 expression was the most significant.

[0159] Figure 31 To knock down the protein expression level of MMP2 in gastric signet ring cell carcinoma cells after PTGS2, Figure 32 To knock down the protein expression level of MMP2 in gastric signet ring cell carcinoma cells after IQGAP1. Figure 31 and Figure 32 It was found that knocking down PTGS2 and IQGAP1 both inhibited the protein expression level of MMP2 in gastric signet ring cell carcinoma cells. Figure 33 To reduce the phosphorylation level of the ERK pathway in gastric signet ring cell carcinoma cells after knocking down IQGAP1, knocking down IQGAP1 inhibited the phosphorylation level of the ERK pathway. Figure 34 To determine the phosphorylation level of the ERK pathway in gastric signet ring cell carcinoma cells after PTGS2 overexpression, PTGS2 overexpression promoted ERK pathway phosphorylation, while the total ERK protein expression level did not change significantly. This suggests that the PTGS2-IQGAP1 axis may promote the development and progression of gastric signet ring cell carcinoma by activating the ERK pathway and upregulating MMP2 expression.

[0160] 2.7 RNA recovery efficiency

[0161] Gastric signet ring cell carcinoma tissue was collected, and experiments were performed according to steps S1 and S2 of the detection methods for gastric signet ring cell carcinoma biomarkers in Examples 1-5 and Comparative Example 1, respectively, to obtain RNA solutions and purified RNA solutions. The concentration of RNA in the RNA solutions and purified RNA solutions was detected using a Nanodrop micro-spectrophotometer. The concentration of RNA in the RNA solution was denoted as C1, and the volume of the RNA solution was denoted as V1; the concentration of RNA in the purified RNA solution was denoted as C2, and the volume of the purified RNA solution was denoted as V2. RNA recovery efficiency (%) = (C2 × V2) / (C1 × V1) × 100%. Table 1 shows the RNA recovery efficiency (%).

[0162] Table 1. RNA recovery efficiency (%)

[0163]

[0164] As shown in Table 1, the RNA recovery efficiency of Examples 2-3 of this invention is higher than that of Example 1. This is because the magnetic bead suspensions used in Examples 1 and 2-3 are different. In the preparation of the magnetic bead suspension, Example 1 uses oleic acid-modified magnetite particles reacting with ethyl silicate to obtain the magnetic bead suspension. In Examples 2-3, oleic acid-modified magnetite particles are first reacted with ethyl silicate, and then reacted with diethylenetriaminepropyltrimethoxysilane and 5-chloro,4-methylpentanoyl chloride to obtain the magnetic bead suspension. The RNA recovery efficiency of Examples 2-3 is higher than that of Comparative Example 1 because 5-chloro,4-methylpentanoyl chloride was not used in the preparation of the magnetic bead suspension in Comparative Example 1. The RNA recovery efficiency of Example 2 is higher than that of Example 3 because the amount of magnetic bead suspension used in Examples 2 and 3 is different. This indicates that the magnetic bead suspension prepared by this invention can efficiently purify RNA and improve the RNA purification and recovery efficiency.

[0165] The RNA recovery efficiency of Examples 4-5 is higher than that of Example 2 because the magnetic bead suspensions used in Examples 2 and 4-5 are different. In the preparation of the magnetic bead suspension, Examples 4-5 use oleic acid-modified magnetite particles that are first reacted with ethyl silicate, then with diethylenetriaminepropyltrimethoxysilane and 5-chloro,4-methylpentanoyl chloride, and finally with N-(tert-butoxycarbonyl)ethanolamine to obtain the magnetic bead suspension. Examples 2-3 use oleic acid-modified magnetite particles that are first reacted with ethyl silicate, then with diethylenetriaminepropyltrimethoxysilane and 5-chloro,4-methylpentanoyl chloride to obtain the magnetic bead suspension. The RNA recovery efficiency of Examples 4-5 is higher than that of Comparative Example 2 because N-(tert-butoxycarbonyl)ethanolamine was not used in the preparation of the magnetic bead suspension in Comparative Example 2. The RNA recovery efficiency of Example 4 is higher than that of Example 5 because the amount of magnetic bead suspension used in Examples 4 and 5 is different. This demonstrates that the magnetic bead suspension prepared in Example 4 of this invention can further improve the efficiency of RNA purification and recovery.

[0166] 2.8 Specificity test of the detection kit for gastric signet ring cell carcinoma biomarkers

[0167] Gastric signet ring cell carcinoma tissues were collected and tested according to the detection methods for gastric signet ring cell carcinoma biomarkers in Examples 1-5 and Comparative Example 1. Each experiment was performed with 4 biological replicates and 4 technical replicates. Specificity (%) = number of positive experimental groups / total number of experimental groups × 100%. Table 2 shows the specificity (%).

[0168] Table 2 Specificity (%)

[0169]

[0170] As shown in Table 2, the specificity of the detection kits for gastric signet ring cell carcinoma biomarkers in Examples 2-3 of this invention is higher than that in Example 1 and Comparative Example 1. This is because the magnetic bead suspensions used in Examples 1, Comparative Example 1, and Examples 2-3 are different. The magnetic bead suspensions prepared in Examples 2-3 can efficiently purify RNA, reverse transcribe high-purity RNA into cDNA, and then use cDNA as a template for qRT-PCR experiments, reducing the influence of impurities on qRT-PCR, thereby improving the specificity of the detection kits for gastric signet ring cell carcinoma biomarkers of this invention. The specificity of the detection kit for gastric signet ring cell carcinoma biomarkers in Example 2 is higher than that in Example 3, because the amount of magnetic bead suspension used in Examples 2 and 3 is different. This indicates that the detection kits for gastric signet ring cell carcinoma biomarkers of this invention have excellent specificity.

[0171] The specificity of the gastric signet ring cell carcinoma biomarker detection kits in Examples 4-5 of this invention is higher than that in Examples 2 and Comparative Example 2. This is because the magnetic bead suspensions used in Examples 2, Comparative Example 2, and Examples 4-5 are different. The magnetic bead suspensions prepared in Examples 4-5 can further optimize the purification and recovery efficiency of RNA, reverse transcribe high-purity RNA into cDNA, and then use cDNA as a template for qRT-PCR experiments, reducing the influence of impurities on qRT-PCR, thereby further optimizing the specificity of the gastric signet ring cell carcinoma biomarker detection kits of this invention. The specificity of the gastric signet ring cell carcinoma biomarker detection kit in Example 4 is higher than that in Example 5 because the amount of magnetic bead suspension used in Examples 4 and 5 is different. This indicates that the present invention, by using the magnetic bead suspension prepared in Example 4 to compose the gastric signet ring cell carcinoma biomarker detection kit, can further optimize the specificity of the gastric signet ring cell carcinoma biomarker detection kit.

[0172] The conventional operations in the operation steps of this invention are well known to those skilled in the art and will not be described in detail here.

[0173] The embodiments described above provide a detailed explanation of the technical solutions of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any changes or modifications made within the scope of the principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A detection kit for biomarkers of gastric signet ring cell carcinoma, comprising, Total RNA extraction reagent, RNA purification reagent, cDNA first-strand synthesis premixed reagent, rapid real-time fluorescence premixed reagent SYBR Green, nuclease-free double-distilled water, and specific primers; The RNA purification reagent includes isopropanol, chloroform, anhydrous ethanol, and a magnetic bead suspension; the specific primers include an upstream specific primer and a downstream specific primer, the nucleotide sequence of the upstream specific primer is 5'-TTCTCGTGAAGCCCTATGA-3', and the downstream specific nucleotide sequence is 5'-GAGGCAGTGTTGATGATTTT-3'; the specific method for preparing the magnetic bead suspension is as follows: under a nitrogen atmosphere, oleic acid-modified iron oxide magnetic particles are added to deionized water and stirred and dispersed; ammonia water is added and stirred and dispersed again; ethyl silicate is added dropwise. The ester was stirred and reacted at 35-50℃ for 1-3 hours. After the reaction was completed, it was naturally cooled to room temperature. Ammonia was added to adjust the pH to 9-10. Diethylenetriaminepropyltrimethoxysilane was added and reacted for 4-8 hours. 5-chloro,4-methylpentanoyl chloride and triethylamine were added at 0-5℃ and stirred and reacted at room temperature for 3-6 hours. N-(tert-butoxycarbonyl)ethanolamine, anhydrous potassium carbonate and potassium iodide were then added and reacted at 70-80℃ for 2-6 hours. After the reaction was completed, it was naturally cooled to room temperature. It was washed with anhydrous ethanol until the pH was neutral. After magnetic separation, ethanol solution was added and ultrasonically dispersed to obtain a magnetic bead suspension.

2. The detection kit for gastric signet ring cell carcinoma biomarkers according to claim 1, characterized in that, The biomarker for gastric signet ring cell carcinoma is prostaglandin intraepoxide synthase 2.

3. The detection kit for gastric signet ring cell carcinoma biomarkers according to claim 1, characterized in that, The ratio of oleic acid-modified iron oxide magnetic particles to ammonia is 1g:5-20mL.

4. The detection kit for gastric signet ring cell carcinoma biomarkers according to claim 1, characterized in that, The ratio of oleic acid-modified iron oxide magnetic particles to ethyl silicate is 1g:2-10mL.

5. The detection kit for gastric signet ring cell carcinoma biomarkers according to claim 1, characterized in that, The ratio of oleic acid-modified iron oxide magnetic particles to diethylenetriaminepropyltrimethoxysilane is 1g:2-10mL.

6. The detection kit for gastric signet ring cell carcinoma biomarkers according to claim 1, characterized in that, The mass ratio of the oleic acid-modified magnetite particles and 5-chloro,4-methylpentanoyl chloride is 1:2-10.

7. The detection kit for gastric signet ring cell carcinoma biomarkers according to claim 1, characterized in that, The ethanol solution has a mass concentration of 75-95%.

8. The detection kit for gastric signet ring cell carcinoma biomarkers according to claim 1, characterized in that, The ratio of oleic acid-modified iron oxide magnetic particles to ethanol solution is 1g:50-150mL.

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